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Genes to Cells (2008) 13, 1-12. doi:10.1111/j.1365-2443.2007.01145.x
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Functional dissection of transformation by c-Src and v-Src

 Chitose Oneyama, Tomoya Hikita, Shigeyuki Nada and Masato Okada*

Department of Oncogene Research, Research Institute of Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The c-src proto-oncogene product, c-Src, is frequently over-expressed and activated in various human malignant cancers, implicating a role for c-Src in cancer progression. To verify the role of c-Src, we analyzed the transforming ability of c-Src in mouse embryonic fibroblasts that lack Csk, a negative regulator of Src family kinases. Although Csk deficiency is not sufficient for cell transformation, c-Src over-expression induced characteristic transformed phenotypes including anchorage-independent growth and tumorigenecity. These phenotypes were dose-dependently inhibited by the re-expression of Csk, indicating that there is a certain threshold for c-Src transformation, which is determined by the c-Src : Csk ratio. In contrast to v-Src, c-Src induced the phosphorylation of a limited number of cellular proteins and elicited a restricted change in gene expression profiles. The activation of some critical targets for v-Src transformation, such as STAT3, was not significantly induced by c-Src transformation. Several genes that are involved in cancer progression, that is, cyclin D1 and HIF-1{alpha}, were induced by v-Src, but not by c-Src. Furthermore, v-Src tumors exhibited aggressive growth and extensive angiogenesis, while c-Src tumors grew more slowly accompanied by the induction of hematomas. These findings demonstrate that c-Src has the potential to induce cell transformation, but it requires coordination with an additional pathway(s) to promote tumor progression in vivo.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
c-Src, a non-receptor tyrosine kinase, was originally identified as the cellular prototype of v-Src, an oncogene product of Rous sarcoma virus (Takeya & Hanafusa 1983; Brown & Cooper 1996). As c-Src is over-expressed and activated in a wide variety of human cancers, particularly colon and breast cancers, much attention has been focused on its potential involvement in the etiology of malignant conversion in human cancers (Irby & Yeatman 2000; Ishizawar & Parsons 2004; Yeatman 2004). The c-src gene is rarely mutated in human cancers, except for a small subset of cancers including some colon cancers and c-Src over-expression in normal cells scarcely induces transformation (Irby et al. 1997; Biscardi et al. 2000; Ishizawar & Parsons 2004). Numerous studies have defined the normal functions of c-Src in regulating cell adhesion and migration as well as in growth factor signaling (Frame 2002, 2004; Yeatman 2004), but the critical pathways linked to cancer progression still remain to be elucidated. As c-Src has little or no transforming activity in normal cells, most studies of Src-induced transformation have been performed with v-Src or a c-Src mutant lacking the negative regulatory site (Irby et al. 1999). The structure of v-Src differs from that of c-Src in that v-Src lacks the regulatory C-terminal domain of c-Src and has several point mutations, both of which coordinately render v-Src constitutively active and confer a high transformation activity (Takeya & Hanafusa 1982; Jove & Hanafusa 1987; Yeatman 2004). Cellular transformation by these active forms of Src results in the tyrosine phosphorylation of a wide variety of cellular proteins including FAK (Schaller et al. 1992; Hanks & Polte 1997), Cas/Crk (Sakai et al. 1994; Honda et al. 1998), Shc (Verderame et al. 1995) and STAT3 (Yu et al. 1995). However, it remains unclear whether these pathways could also be directly activated by endogenous c-Src during cancer progression.

It is well established that c-Src is negatively regulated by the phosphorylation of its C-terminal regulatory site by the C-terminal Src Kinase, Csk (Nada et al. 1991; Roskoski 2004) and that Csk-mediated negative regulation is essential for the development and homeostasis in animals (Nada et al. 1993; Cole et al. 2003). Given that Csk expression is reduced in some human cancers (Masaki et al. 1999; Cam et al. 2001), it is possible that the down-regulation of Csk is involved in the up-regulation of c-Src activity. In contrast, an increase in c-src gene expression and the stabilization of c-Src protein, as observed in certain breast cancer cells (Tan et al. 2005), also provides another mechanism for c-Src up-regulation. In either case, disequilibrium of the c-Src : Csk ratio would be expected to contribute, at least partly, to the up-regulation of c-Src activity, although there has been no experimental evidence that supports this possibility.

To verify the function of c-Src in cancer progression, we developed an experimental system using Csk-deficient mouse embryonic fibroblasts (Csk–/– MEF), in which c-Src could induce cell transformation under the control of Csk. This system enabled us to compare the transforming ability of c-Src with that of v-Src. Our results show that c-Src can activate cellular pathways essential for in vitro cell transformation, but, unlike v-Src, it does not remarkably induce tumor progression in vivo. The functional difference between c-Src and v-Src is further investigated by biochemical and DNA microarray analyses.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
c-Src induces transformation of Csk-deficient fibroblasts

To examine whether wild-type c-Src could be involved in cell transformation, we introduced c-Src into normal MEFs (Csk+/+) and Csk–/– MEFs (Imamoto & Soriano 1993). In the presence of Csk (Csk+/+), the over-expression of c-Src did not affect the cell morphology (Fig. 1A, b) or induce colony formation activity (Fig. 1B, b), indicating that c-Src activity is strictly regulated by endogenous Csk (Ishizawar & Parsons 2004). The expression of the constitutively active form of c-Src (c-SrcYF), which has a Tyr to Phe replacement in its regulatory site Tyr527, could induce morphological changes (Fig. 1A, c) as well as colony formation activity (Fig. 1B, c) in Csk+/+ cells. As previously observed (Nada et al. 1994), Csk–/– MEFs showed some changes in morphology due to the activation of endogenous Src family kinases (Fig. 1A, d). However, these cells did not show significant colony formation activity (Fig. 1B, d), indicating that activation of endogenous c-Src is insufficient for cell transformation. This could be explained by the down-regulation of activated c-Src proteins (Fig. 2A, Src blots, lanes 4 and 5) through the ubiquitin-dependent pathway (Hakak & Martin 1999). When c-Src was over-expressed in Csk–/– MEFs (Csk–/–/c-Src), the cells became smaller, disorganized and refractile, characteristic of a transformed phenotype (Fig. 1A, e). Furthermore, c-Src expression in these cells induced strong colony formation activity comparable to that induced by c-SrcYF or v-Src (Fig. 1B, e). These transformed phenotypes were completely suppressed by the expression of wild-type Csk (Fig. 1A, h and B, i), but not by its kinase-negative mutant (CskKN) (Fig. 1A, i and B, j). These observations demonstrate that c-Src activity in Csk–/–/c-Src cells is sufficient for cell transformation and that the intrinsic transforming ability of c-Src is suppressed by Csk in normal cells.


Figure 1
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  		Figure 1  c-Src induces the transformation of Csk-deficient fibroblasts. (A) Csk+/+ or Csk–/– fibroblasts were infected with retrovirus expressing c-Src (b and e), the oncogenic form of c-Src (c-SrcYF) (c and f) or the control vector (Mock) (a and d). Csk–/– cells expressing c-Src (Csk–/–/c-Src) were infected with retrovirus expressing Csk (h), kinase-deficient Csk (CskKN) (i) or the control vector (Mock). Cell morphologies were examined by phase-contrast microscopy at 100x magnification. (B) Soft agar colony-formation assays of Csk+/+ or Csk–/– fibroblasts expressing the indicated genes. Colonies were stained with MTT and photographed 2 weeks after plating. Representative dishes from three independent experiments are shown. The number of stained colonies was counted for Csk–/– fibroblasts expressing c-Src, c-SrcYF, v-Src and Csk or mock; the mean and standard deviations obtained in three independent experiments are given. (C) Immunocytochemical analysis of c-Src and v-Src transformed cells. Indicated cell lines were co-stained with Alexa594-phalloidin (red) and 4G10 (green). Other specimens were stained with anti-cortactin (green).

 

Figure 2
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  		Figure 2  The balance between c-Src and Csk determines c-Src-induced transformation. (A) Total cell lysates from Csk+/+ or Csk–/– fibroblasts used in Fig. 1B were subjected to immunoblot analysis with the anti-phosphotyrosine antibody 4G10, anti-Src, anti-Src pY416, anti-Src pY527 or anti-Csk. The increase in the phosphorylation of Src pY527 in Csk–/–/c-SrcYF cells is due to the phosphorylation of endogenous c-Src Y527, which can be performed by c-SrcYF. (B) c-Src was expressed in Csk–/– fibroblasts and cells expressing different levels of c-Src were cloned (c-Src, clones 1–5, lanes 1–5). The cells that expressed the highest levels of c-Src (c-Src 5) were then infected with a retrovirus expressing Csk and several clones expressing different levels of Csk were isolated (c-Src5/Csk, clones 1–4, lanes 6–9). Src and Csk were simultaneously expressed in Csk–/– fibroblasts and the cells expressing different ratios of c-Src to Csk were cloned (c-Src/Csk, clones 1–5, lanes 10–14). The relative ratio of expression of c-Src to Csk is shown at the bottom of the panels. These cells were subjected to immunoblot analysis with the antibodies indicated on the right. (C) The ability of the cell clones used in (B) for anchorage-independent growth was analyzed using the soft agar colony-formation assay. The clone number is indicated at the top of each panel. Colonies were stained with MTT and photographed 2 weeks after plating.

 
The cellular architecture of c-Src, v-Src or c-SrcYF transformed cells was then examined by staining for F-actin, tyrosine phosphorylated proteins and cortactin (Fig. 1C). Cell transformation by c-Src induced a dramatic accumulation of F-actin, tyrosine phosphorylated proteins and cortactin at the podosome, a characteristic structure of Src-transformed cells that is implicated in cancer invasiveness (+c-Src panels). Reintroduction of Csk into these cells almost completely restored the normal cytoskeletal organization (+c-Src/Csk). v-Src transformation also affected the cytoskeleton, but the actin organization was more severely disrupted and the size of the podosomes was reduced (+v-Src). Similar effects were observed with c-SrcYF, except for the relatively large size of the podosomes (+c-SrcYF). These observations show that c-Src activation is sufficient for inducing cytoskeletal remodeling characteristic of cell transformation, although it is less effective than v-Src or c-SrcYF.

The balance between c-Src and Csk determines c-Src transformation

We next compared the cellular events induced by c-Src, c-SrcYF or v-Src transformation. The tyrosine phosphorylation of cellular proteins was greatly elevated in c-SrcYF or v-Src transformed cells, irrespective of the presence of Csk (Fig. 2A, pY blots, lanes 3, 7 and 10). In contrast, c-Src transformed Csk–/– cells (Csk–/–/c-Src) contained a relatively small number of phosphorylated proteins compared to c-SrcYF or v-Src transformed cells (Fig. 2A, pY blots, lanes 6 and 9), even though c-Src was substantially activated as judged by its elevated autophosphorylation at pY416 (Fig. 2A, pY416 blots, lanes 6 and 9). Re-expression of wild-type Csk suppressed protein phosphorylation in Csk–/–/c-Src cells, but not in c-SrcYF transformed cells (Fig. 2A, lanes 12 and 15). These results suggest that there is a qualitative difference in the function of c-Src and v-Src and that phosphorylation of a relatively limited number of target proteins is sufficient for c-Src transformation.

In order to determine the degree of c-Src activity that is required for cell transformation, we subcloned Csk–/– cell lines expressing different levels of c-Src and Csk proteins. In the Csk–/–/c-Src cells, the c-Src expression level was tightly correlated with the levels of protein tyrosine phosphorylation (Fig. 2B, pY blots) and c-Src activity (Src pY416), as well as with colony formation activity (Fig. 2C, upper panels), indicating that cell transformation is dependent on c-Src activity. Notably, a two- to threefold increase in the c-Src protein level was sufficient to induce cell transformation in these cells. We then re-expressed Csk into a subclone of Csk–/–/c-Src cells that expressed the highest level of c-Src (c-Src5)) and isolated several clones that exhibited different levels of Csk (c-Src5/Csk). The transformed phenotypes, including colony formation activity, were substantially suppressed depending upon the level of Csk (Fig. 2B, lanes 6–9 and C, middle panels). Even in clones expressing relatively high levels of Csk (Csk–/–/c-Src/Csk), the c-Src : Csk ratio appeared to correlate with the severity of the transformed phenotypes (Fig. 2B, lanes 10–14 and C, bottom panels). These results suggest that cell transformation can be induced when c-Src activity is elevated above a certain threshold and that a disturbance in the c-Src : Csk ratio could contribute to the induction of cell transformation.

Tumor formation induced by c-Src and v-Src

To characterize c-Src transformed cells in vivo, a series of Csk–/– cell derivatives were injected subcutaneously into nude mice. v-Src or c-SrcYF transformed cells grew aggressively and formed large tumors within 2 weeks (Fig. 3A). In contrast, c-Src transformed cells required more than 3 weeks to form tumors that were comparable to those produced by v-Src transformed cells (Fig. 3B). The introduction of Csk into Csk–/–/c-Src cells substantially suppressed tumor formation and Csk–/– cells did not form any detectable tumors within 3 weeks. Furthermore, we observed that v-Src tumors promoted angiogenesis much more extensively than c-Src tumors (Fig. 3C, a and b), while c-Src tumors induced the formation of massive hematomas (Fig. 3C, c and d). These observations suggest that there are functional differences between c-Src and v-Src or c-SrcYF in vivo.


Figure 3
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  		Figure 3  Tumorigenicity of c-Src transformed cells in vivo. (A) Locations injected with Csk–/– cells expressing v-Src/Csk (a), control vector (b), c-SrcYF/Csk (c), c-Src (d) and c-Src/Csk (e) are indicated by arrowheads. (B) Mean and standard deviation of the tumor volume (mm3) of groups of five mice injected with Csk–/– cells expressing v-Src/Csk (black squares), c-Src (black circles), c-Src/Csk (white circles), c-SrcYF/Csk (black triangles) or the vector (Mock; white diamonds) vs. time (days). (C) Cross-section of tumors derived from Csk–/–/v-Src/Csk (a) or Csk–/–/c-Src (c) cells. Tumor sections derived from Csk–/–/v-Src/Csk (b) or Csk–/–/c-Src (d) cells were subjected to H&E staining and observed under phase-contrast microscopy at 400x magnification.

 
Signal transduction induced by c-Src and v-Src transformation

In order to characterize the signaling pathways that are activated by c-Src transformation, we examined the phosphorylation of some Src substrates that were previously identified in v-Src transformed cells (Yeatman 2004). Phosphorylation of FAK, Shc and cortactin was increased in c-Src transformed cells, although their phosphorylated levels were lower than those in c-SrcYF or v-Src transformed cells (Fig. 4A, lanes 3 and 9). As these proteins were also phosphorylated in transformation defective Csk–/– cells (lanes 1 and 7), it seems likely that the phosphorylation of these proteins is not necessarily sufficient for cell transformation. Furthermore, we observed that c-Src could induce the activation of ERK and AKT, both of which are known to contribute to v-Src transformation (Penuel & Martin 1999), to the levels similar to those in v-Src transformed cells (Fig. 4B). These results show that the basic pathways involved in cell adhesion, proliferation and survival are commonly activated by c-Src and v-Src. In contrast, we found that STAT3, another critical component of v-Src transformation (Bromberg et al. 1999; Schlessinger & Levy 2005), was phosphorylated in c-SrcYF or v-Src transformed cells, but not in c-Src transformed cells (Fig. 4A, lane 3). Even in Src5 cells having the highest expression of c-Src, STAT3 phosphorylation was substantially lower than that in v-Src transformed cells (data not shown). These observations suggest that the STAT3 pathway may not be involved in c-Src transformation.


Figure 4
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  		Figure 4  The cellular events induced by c-Src and v-Src transformation. (A) Phosphorylation of Src substrates. Total cell lysates from Csk–/– fibroblasts expressing the indicated genes were subjected to immunoblot analysis with antibodies to the indicated proteins. (B) The activity status of ERK and AKT. Total cell lysates used in (A) were probed with the indicated antibodies.

 
Contribution of STAT3 to Src-induced transformation and tumorigenesity

To evaluate the role of STAT3 in the differential function of c-Src and v-Src, we introduced the wild-type and a constitutively active form of STAT3 (Bromberg et al. 1999) into c-Src transformed Csk–/– cells (Fig. 5A). Both forms of STAT3 were activated in these cells, without affecting the overall tyrosine phosphorylation levels (Fig. 5A, lanes 2 and 3). The constitutive STAT3 activation could induce only a small change in the cell morphology; the cells tended to exhibit a spindle-shaped morphology resembling v-Src transformed cells (Fig. 5B). As was the case for v-Src transformation, the STAT3 activation did not affect the colony formation activity in soft-agar (Fig. 5B). However, a tumorigenesis assay in nude mice showed that the constitutive activation of STAT3 significantly enhanced tumor formation of c-Src transformed cells, although the cells grew slower than v-Src transformed cells (Fig. 5C). These observations suggest that STAT3 activation could indeed contribute to tumor progression and that STAT3 may be at least one of the components that account for the functional difference between c-Src and v-Src.


Figure 5
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  		Figure 5  The effects of STAT3 activation on c-Src transformed phenotypes. (A) Csk–/– cells expressing c-Src (c-Src) were infected with retrovirus expressing wild-type or constitutively active STAT3 (STAT3CA) and total cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Cell morphologies were photographed at 200x magnification (upper panels) and transforming activity was analyzed using the soft-agar colony formation assay (lower panels). (C) Csk–/– cells expressing c-Src (black circles), c-Src/STAT3CA (black diamonds) or v-Src/Csk (black squares) were injected s.c. into nude mice. Averages ± SD of the tumor volume (mm3) obtained from four mice are plotted vs. time (days). *P < 0.01, by Student's t-test. (D) Phosphorylation of STAT3 in various human cancer cells. Total cell lysates from the human cell lines, MCF7, T-47D or MDA-MB-231 (breast cancer cell line), HT29, HCT116, SW480 or Caco-2 (colon cancer cell line), HaCaT (keratinocyte cell line) and HEK293 (kidney epithelial cell line), were analyzed by immunoblotting with the indicated antibodies.

 
To further investigate the correlation between STAT3 and c-Src in cancer cells, the activity status of STAT3 was examined in several human breast and colon cancer cell lines in which c-Src has been shown to be over-expressed and/or activated (Irby & Yeatman 2000). As shown in Fig. 5D, STAT3 activation (pY705) was not correlated with c-Src activity (pY416), suggesting that STAT3 activation is not necessarily linked to the c-Src pathway in these human cancer cells.

Microarray analysis of c-Src and v-Src transformation

To gain further insights into the downstream mechanisms regulating c-Src transformation, we performed gene expression profiling using a DNA microarray. We first compared parental Csk–/– cells and Csk–/–/c-Src cells (Fig. 6, panel A). Under the experimental conditions employed (P value < 0.01 and fold change > 2.0), only 85 (0.2%) and 58 (0.2%) genes out of 37 290 genes were significantly up- and down-regulated by c-Src transformation, respectively. The reintroduction of Csk into Csk–/–/c-Src cells also caused the up- and down-regulation of genes (panel B), with frequencies of 0.2% and 0.1%, respectively. In contrast, v-Src transformation up- and down-regulated 244 (0.6%) and 411 (1.1%) genes, respectively (panel C), indicating that v-Src affects a much wider range of genes than c-Src.


Figure 6
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  		Figure 6  Gene expression profiling analysis of c-Src and v-Src transformed cells. Total RNA was isolated from Csk–/– cells (Mock), Csk–/– cells expressing c-Src (c-Src), c-Src plus Csk (c-Src/Csk), Csk (Csk) or v-Src plus Csk (v-Src/Csk) and was subjected to micro array gene expression analysis. Gene expression profiles were compared between Mock and c-Src (A), between c-Src and c-Src/Csk and between v-Src/Csk and Csk (C), to identify the genes that were up- or down-regulated by Src activity. The log10 values of the expression intensities for individual genes in each set of cell types indicated were plotted on the x and y axes. Genes showing significant (P < 0.01 and fold change > 2.0) up-regulation and down-regulation are colored red and green, respectively. Red lines indicate a fold change of ± 2.0.

 
It has been shown that c-Src and v-Src can modulate the expression of similar sets of genes (Malek et al. 2002; Paz et al. 2004; Sung et al. 2005; Shen et al. 2006), including matrix-related proteins (collagen and Timp3), transcription factors (Ets), anti-apoptotic proteins (Clu), adaptor proteins (Igfbp3) and substrate proteins (MARCKS and Eps8). Alterations in the expression of these genes by c-Src and v-Src were also detected under our conditions, but the fold changes induced by c-Src were lower than those induced by v-Src (Table 1). This suggests that differences in the amplitude of gene expression induced by c-Src and v-Src may be responsible for their functional differences. The expression of several other proteins such as the cell cycle protein Cyclin D1, the angiogenic protein HIF-1{alpha}, the growth arrest protein Gas1 and the matrix protein fibronectin, were more selectively affected by v-Src (Table 1). Given the importance of HIF-1{alpha} in cancer progression and angiogenesis (Semenza 2003), this observation suggests that the poor vascularization of c-Src tumors may be linked to low levels of HIF-1{alpha} induction. As STAT3 contributes to the induction of Cyclin D1, the low level of Cyclin D1 induction by c-Src may be the result of inefficient STAT3 activation (Fig. 4A). In contrast, c-Src specifically altered the expression of other proteins such as Cpxm1 and Lgi2 (Table 1), although their functions in transformation are unknown. These array data suggest that the basic pathways for cell transformation are commonly activated by c-Src and v-Src, but that additional pathways leading to cancer progression, such as STAT3-mediated angiogenesis, are more potently activated by v-Src.


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  		Table 1  Microarray analysis of representative genes regulated by c-Src or v-Src
 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Using Csk-deficient cells, we have shown that c-Src has an intrinsic transformation activity whose potential depends on the cellular c-Src : Csk ratio. This observation verifies the fact that Csk is a critical suppressor of the transforming potential of c-Src and that destruction of the c-Src/Csk balance could be partially responsible for cell transformation. The in vitro transformed phenotypes that were induced by c-Src appeared almost comparable with those induced by v-Src, except that the cytoskeletal organization was more severely disrupted by v-Src than c-Src. We have also shown that c-Src induced the phosphorylation of a relatively small number of cellular proteins compared to v-Src or c-SrcYF. This may be due to the lower level of c-Src specific activity in Csk-deficient cells, in which a proportion of c-Src can be inactivated by the autophosphorylation of Y527 (Fig. 2A, lane 9, pY527 blots). Alternatively, it is also possible that the substrate specificity of c-Src is different from that of v-Src or c-SrcYF, although more detailed biochemical analysis should be performed to verify this possibility. In either case, the lower levels of phosphorylation in c-Src transformed cells suggest that the activation of restricted pathways is sufficient for in vitro cell transformation. It has been shown that Csk-deficient cells also exhibited an increase in the phosphorylation of some cellular proteins, which was caused by the activation of endogenous Src family kinases (Nada et al. 1994). However, the level of phosphorylation was lower than that in c-Src transformed cells and was insufficient to induce cell transformation. These findings suggest that a comparative analysis of the phosphorylated protein profiles of Csk-deficient cells and c-Src transformed cells would enable us to identify the minimal sets of c-Src target proteins that are required for cell transformation. Thus, our experimental system using Csk-deficient cells would be beneficial for studies on the molecular mechanism of c-Src transformation.

We also found that there was a significant difference in in vivo tumorigenesis between c-Src and v-Src transformed cells. v-Src transformed cells grew aggressively accompanied by extensive angiogenesis, while c-Src transformed cells grew substantially slower and induced the formation of massive hematomas. Biochemical and microarray analyses revealed that some critical signaling components that have been implicated in the promotion of angiogenesis, such as STAT3 (Niu et al. 2002), cyclin D1 and HIF-1{alpha} (Semenza 2003), are not activated in c-Src transformed cells. Although the reasons for hemorrhaging in c-Src tumors are currently unclear, the absence of angiogenesis in c-Src-induced tumors might be associated with the lack of activation of these pathways. The previous observations that the inhibition of STAT3 signaling prevented v-Src transformation (Bromberg et al. 1998; Schlessinger & Levy 2005) demonstrated the critical role for STAT3 in v-Src mediated tumor progression. In this study, we observed that the expression of a constitutively active form of STAT3 could promote the in vivo growth of c-Src transformed cells, supporting our notion that the lack of STAT3 activation in c-Src transformed cells would be responsible for their inefficient tumor progression in vivo. Furthermore, we found that the activity of STAT3 was not correlated with the activity status of c-Src in several human cancer cells, indicating that the STAT3 pathway is not necessarily linked to the c-Src pathway. These observations are consistent with the previous claim that the activation of c-Src could play role in cell transformation, but it is not sufficient for tumor progression, in particular metastasis and angiogenesis (Irby et al. 1997). In contrast, a line of evidence has shown that inhibition of Src activity could attenuate angiogenesis as well as the metastatic potential of cancer cells (Summy & Gallick 2003; Ischenko et al. 2007). These results provide the positive role for c-Src activity in tumor progression. Taken together with our observations, it is thus likely that c-Src is required but not sufficient for tumor progression and that it should play roles by cooperating with an additional pathway(s) that is independently up-regulated in cancers, such as EGF receptor signaling (Biscardi et al. 2000; Ishizawar & Parsons 2004) or STAT3 signaling (Yu et al. 1995; Niu et al. 2002).

c-Src is known as a pivotal component of multiple signaling pathways that regulate proliferation, survival, cell-adhesion and migration, most of which are tightly associated with tumor progression (Frame 2004). It is thus possible that an aberrant up-regulation of upstream components of these pathways would converge on c-Src up-regulation, thereby resulting in the induction of cell transformation and subsequent tumor progression. In this study, we presented an auxiliary role for c-Src in tumor progression. However, the pivotal role of c-Src in regulating a wide variety of signaling pathways suggests that c-Src still represents a central therapeutic target for various types of cancer.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cells and cell culture

Csk–/– and sibling Csk+/+ mouse embryo fibroblasts immortalized using the simian virus 40 large T antigen were a kind gift from Dr Akira Imamoto (Thomas et al. 1995) and were cultured in DMEM supplemented with 10% FBS.

Retroviral-mediated gene transfer

All gene transfer experiments were carried out using retroviral vectors at a high efficiency (> 80%), as estimated by the use of a GFP-expressing vector. Retroviral vectors encoding wild-type chicken c-Src, its active form (c-SrcYF) and v-Src were kindly provided by Dr Tsuyoshi Akagi (Osaka Bioscience Institute, Osaka). Wild-type rat Csk and its kinase-deficient mutant (K222R) were subcloned into a retroviral vector CX4bleo. Wild-type STAT3 and its constitutively active mutant (A662C, N664C) were kindly provided by Dr Toru Ouchi (Northwestern University, Evanston, IL) and subcloned into CX4bleo. The production of retroviral vectors and their infection weas performed as described previously (Akagi et al. 2003).

Immunochemical analysis

Immunoblotting was performed as described (Segawa et al. 2006). The following antibodies were used: anti-phosphotyrosine (4G10, Upstate), anti-Src (Ab-1, Oncogene Research Products, San Diego, CA), anti-Src pY418 (BIOSOURCE), anti-Src (BIOSOURCE, Carlsbad, CA), anti-Csk (Santa Cruz, Santa Cruz, CA), anti-FAK pY397 (BIOSOURCE), anti-FAK (Santa Cruz), anti-Stat3 pY705 (Cell Signaling, Beverly, MA), anti-Stat3 (Cell Signaling), anti-Shc pY239/240 (BIOSOURCE), anti-Shc (Transduction Laboratory, Lexington, KY), anti-Cortactin pY421 (BIOSOURCE) and anti-cortactin (Upstate, Lake Placid, NY). For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with Tris-buffered saline containing 0.1% Tween20 (TTBS), the samples were blocked with BSA/TTBS, followed by incubation with primary antibodies and Alexa594-phalloidin (for F-actin staining) in TTBS overnight at 4 ºC. After incubation with FITC-conjugated secondary antibodies, cover slips were mounted on glass slides. The specimens were examined by confocal laser-scanning microscopy (EV-1000, Olympus, Tokyo, Japan).

Soft-agar colony-formation assays

Single-cell suspensions of 4 x 104 cells were plated per 60-mm culture dish in 3 mL of DMEM containing 10% FCS and 0.36% agar on a layer of 5 mL of the same medium containing 0.7% agar. Two weeks after plating, colonies were stained with 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) and photographs of the stained colonies were taken.

Microarray gene expression analyses

Total RNA was extracted using Sepasol (Nacalai, Kyoto, Japan) and then hybridized to the Whole Mouse Genome Microarray (Agilent, Santa Clara, CA). The methods for labeling, hybridization, scanning and gene analysis were performed as described previously (Ishii et al. 2005).

Tumorigenisity assays

Immunodeficient mice (BALB/c AJc1-nu/nu, Japan CLEA, Inc.) were subcutaneously injected with 1 x 106 cells suspended in 200 µL of serum-free DMEM at one location. Tumors were monitored every 2 or 3 days and the tumor volume was estimated using the following formula: 0.5 x L x W2. Four or more mice were used in each experiment. The mice used for this study were handled in strict adherence with local governmental and institutional animal-care regulations.


   Acknowledgements
 
We thank Dr D. Okuzaki of the DNA-chip Development Center for Infectious Diseases (RIMD, Osaka University) for microarray analysis and Drs A. Imamoto, T. Akagi and T. Ouchi for generous gifts of reagents. This work was supported by a grant-aid for Scientific Research of Priority Areas, Cancer and for Young Scientist from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Tadashi Yamamoto

* Correspondence: E-mail: okadam{at}biken.osaka-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 3 July 2007
Accepted: 24 September 2007




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